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Membrane tumor necrosis factor (TNF) induced cooperative signaling of TNFR60 and TNFR80 favors induction of cell death rather than virus production in HIV-infected T cells.

Lazdins JK, Grell M, Walker MR, Woods-Cook K, Scheurich P, Pfizenmaier K - J. Exp. Med. (1997)

Bottom Line: In the present study we have demonstrated by means of antagonistic and agonistic receptor-specific antibodies that in latently infected lymphocytic (ACH-2) cells the TNFR60 plays a dominant role in signaling HIV production, although selective activation of TNFR80 by receptor-specific antibodies can also induce HIV production.Unexpectedly, when both TNFRs were activated simultaneously by agonistic antibodies or coculture with cells expressing a noncleavable membrane form of TNF, HIV production was downregulated and induction of cell death was enhanced in ACH-2 cells.These data provide evidence for the differential trigger potential of membrane versus soluble TNF and show that TNFR80 is an important modulator of TNF responsiveness of HIV-infected T cells via cooperative signaling with TNFR60.

View Article: PubMed Central - PubMed

Affiliation: Division Pharma, Ciba, Basel, Switzerland.

ABSTRACT
Tumor necrosis factor (TNF) and lymphotoxin (LT) are highly pleiotropic cytokines that play a central role in regulating HIV-1 replication. These cytokines express their activities through two membrane receptors, TNFR60 (p55-60) and TNFR80 (p75-80). In the present study we have demonstrated by means of antagonistic and agonistic receptor-specific antibodies that in latently infected lymphocytic (ACH-2) cells the TNFR60 plays a dominant role in signaling HIV production, although selective activation of TNFR80 by receptor-specific antibodies can also induce HIV production. Unexpectedly, when both TNFRs were activated simultaneously by agonistic antibodies or coculture with cells expressing a noncleavable membrane form of TNF, HIV production was downregulated and induction of cell death was enhanced in ACH-2 cells. More relevant, in vitro HIV-infected peripheral blood lymphocytes cocultured with cells expressing membrane TNF underwent rapid induction of apoptosis with a subsequent reduced HIV production of these lymphocytes cultures. This was not observed with HIV-infected lymphocytes treated with soluble TNF. These data provide evidence for the differential trigger potential of membrane versus soluble TNF and show that TNFR80 is an important modulator of TNF responsiveness of HIV-infected T cells via cooperative signaling with TNFR60.

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TNFR80 enhances TNF mediated cytotoxicity in ACH-2  cells. ACH-2 cells were cultured in triplicates with TNF (10 ng/ml) in  the presence of the indicated concentrations of the TNFR80-specific agonist M80 (filled circle) or Fab produced from purified M80 IgG (open  square). Shown is one experiment out of a total of four with similar results.  (A) Supernatant RT activity (mean values SD) was determined after 3 d  of culture. (B) The total cell number of ACH-2 was determined by the  FDA method. Data are expressed as the relative cell numbers (100% =  control cells in the absence of TNF, mean values ± SD).
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Figure 5: TNFR80 enhances TNF mediated cytotoxicity in ACH-2 cells. ACH-2 cells were cultured in triplicates with TNF (10 ng/ml) in the presence of the indicated concentrations of the TNFR80-specific agonist M80 (filled circle) or Fab produced from purified M80 IgG (open square). Shown is one experiment out of a total of four with similar results. (A) Supernatant RT activity (mean values SD) was determined after 3 d of culture. (B) The total cell number of ACH-2 was determined by the FDA method. Data are expressed as the relative cell numbers (100% = control cells in the absence of TNF, mean values ± SD).

Mentions: The previous set of experiments had indicated that TNFR-specific agonistic antibodies in combination can induce an ACH-2 cell response pattern distinct from that inducible by TNF. To determine which of the two TNFRs is triggered differentially by natural ligand versus agonistic antibody, we titrated the antibodies into ACH-2 cultures stimulated by a constant concentration of TNF. In all experiments the TNFR60 agonist htr-1 displayed no significant additional effects on either TNFinduced RT induction or growth inhibition (data not shown), whereas the TNFR80 agonist M80 strongly diminished the TNF-induced RT induction and enhanced the TNF-induced cytotoxicity in parallel. Although the quantity of these M80 effects varied considerably within the four experiments performed, they were significant in all cases and could even lead to a total inhibition of RT induction, paralleled by killing of all ACH-2 cells at the end of a three-day culture (Fig. 5). To achieve this activation of TNFR80, complete antibodies were required, as Fab fragments of the agonist M80 showed no effect (Fig. 5). In the presence of the TNFR60 antagonistic antibody H398, not only virus replication (Fig. 2) but also the TNF dependent cell death was fully reverted (data not shown). Together, these data suggest that efficient induction of cell death in ACH-2 requires cooperative signaling via both TNFRs.


Membrane tumor necrosis factor (TNF) induced cooperative signaling of TNFR60 and TNFR80 favors induction of cell death rather than virus production in HIV-infected T cells.

Lazdins JK, Grell M, Walker MR, Woods-Cook K, Scheurich P, Pfizenmaier K - J. Exp. Med. (1997)

TNFR80 enhances TNF mediated cytotoxicity in ACH-2  cells. ACH-2 cells were cultured in triplicates with TNF (10 ng/ml) in  the presence of the indicated concentrations of the TNFR80-specific agonist M80 (filled circle) or Fab produced from purified M80 IgG (open  square). Shown is one experiment out of a total of four with similar results.  (A) Supernatant RT activity (mean values SD) was determined after 3 d  of culture. (B) The total cell number of ACH-2 was determined by the  FDA method. Data are expressed as the relative cell numbers (100% =  control cells in the absence of TNF, mean values ± SD).
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Related In: Results  -  Collection

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Figure 5: TNFR80 enhances TNF mediated cytotoxicity in ACH-2 cells. ACH-2 cells were cultured in triplicates with TNF (10 ng/ml) in the presence of the indicated concentrations of the TNFR80-specific agonist M80 (filled circle) or Fab produced from purified M80 IgG (open square). Shown is one experiment out of a total of four with similar results. (A) Supernatant RT activity (mean values SD) was determined after 3 d of culture. (B) The total cell number of ACH-2 was determined by the FDA method. Data are expressed as the relative cell numbers (100% = control cells in the absence of TNF, mean values ± SD).
Mentions: The previous set of experiments had indicated that TNFR-specific agonistic antibodies in combination can induce an ACH-2 cell response pattern distinct from that inducible by TNF. To determine which of the two TNFRs is triggered differentially by natural ligand versus agonistic antibody, we titrated the antibodies into ACH-2 cultures stimulated by a constant concentration of TNF. In all experiments the TNFR60 agonist htr-1 displayed no significant additional effects on either TNFinduced RT induction or growth inhibition (data not shown), whereas the TNFR80 agonist M80 strongly diminished the TNF-induced RT induction and enhanced the TNF-induced cytotoxicity in parallel. Although the quantity of these M80 effects varied considerably within the four experiments performed, they were significant in all cases and could even lead to a total inhibition of RT induction, paralleled by killing of all ACH-2 cells at the end of a three-day culture (Fig. 5). To achieve this activation of TNFR80, complete antibodies were required, as Fab fragments of the agonist M80 showed no effect (Fig. 5). In the presence of the TNFR60 antagonistic antibody H398, not only virus replication (Fig. 2) but also the TNF dependent cell death was fully reverted (data not shown). Together, these data suggest that efficient induction of cell death in ACH-2 requires cooperative signaling via both TNFRs.

Bottom Line: In the present study we have demonstrated by means of antagonistic and agonistic receptor-specific antibodies that in latently infected lymphocytic (ACH-2) cells the TNFR60 plays a dominant role in signaling HIV production, although selective activation of TNFR80 by receptor-specific antibodies can also induce HIV production.Unexpectedly, when both TNFRs were activated simultaneously by agonistic antibodies or coculture with cells expressing a noncleavable membrane form of TNF, HIV production was downregulated and induction of cell death was enhanced in ACH-2 cells.These data provide evidence for the differential trigger potential of membrane versus soluble TNF and show that TNFR80 is an important modulator of TNF responsiveness of HIV-infected T cells via cooperative signaling with TNFR60.

View Article: PubMed Central - PubMed

Affiliation: Division Pharma, Ciba, Basel, Switzerland.

ABSTRACT
Tumor necrosis factor (TNF) and lymphotoxin (LT) are highly pleiotropic cytokines that play a central role in regulating HIV-1 replication. These cytokines express their activities through two membrane receptors, TNFR60 (p55-60) and TNFR80 (p75-80). In the present study we have demonstrated by means of antagonistic and agonistic receptor-specific antibodies that in latently infected lymphocytic (ACH-2) cells the TNFR60 plays a dominant role in signaling HIV production, although selective activation of TNFR80 by receptor-specific antibodies can also induce HIV production. Unexpectedly, when both TNFRs were activated simultaneously by agonistic antibodies or coculture with cells expressing a noncleavable membrane form of TNF, HIV production was downregulated and induction of cell death was enhanced in ACH-2 cells. More relevant, in vitro HIV-infected peripheral blood lymphocytes cocultured with cells expressing membrane TNF underwent rapid induction of apoptosis with a subsequent reduced HIV production of these lymphocytes cultures. This was not observed with HIV-infected lymphocytes treated with soluble TNF. These data provide evidence for the differential trigger potential of membrane versus soluble TNF and show that TNFR80 is an important modulator of TNF responsiveness of HIV-infected T cells via cooperative signaling with TNFR60.

Show MeSH
Related in: MedlinePlus